Construction of recombinant retrovirus vector carrying hTERT and transfected to MSCs in human cord blood
- VernacularTitle:hTERT-逆转录病毒载体构建及其介导的脐血间质干细胞基因转移研究
- Author:
Ke LI
;
Ruimin LIU
;
Xuefei HAN
;
Ying XING
- Publication Type:Journal Article
- Keywords:
Mesenchymal stem cells;
Cord blood;
Human telomerase reverse transcriptase;
Retrovirus vector
- From:
Chinese Journal of Pathophysiology
1986;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To construct a recombinant retrovirus vector carrying hTERT for establishing UCBMSCs with hTERT(hTERT-MSCs) to overcome their limited life span and detecting whether telomerized UCBMSCs line maintained long-term self-renewal and differentiation capacity.METHODS: The whole cDNA was generated by PCR amplifications from the plasmid pEGFP-hTERT-C1.The hTERT segments were subcloned into pLNCX2.The target cells were infected with these retroviral particles.The stably transfected cells were selected by neomycin and expanded life span which were designated hTERT-MSCs was observed.The expression of hTERT in mRNA level was detected by RT-PCR and the telomerase activity was measured by TRAP(PCR)-ELISA assay.The hTERT-MSCs were induced with 5-azacytidine to cardiac muscle cells and the specific marker of myocardiocyte was detected.RESULTS: The constructed plasmids were digested with restriction endonucleases(BglⅡand NotⅠ).Two characteristic segments including 6.1 kb and 3.6 kb were obtained.The hTERT-MSCs expressed hTERT in mRNA level.The telomerase activity of hTERT-MSCs was positive.The growth kinetics of hTERT-MSCs was higher than those in UCBMSCs.The hTERT-MSCs were induced to myocardiocyte.CONCLUSION: The hTERT recombinant retrovirus vector has been successfully constructed.The hTERT gene activates the telomerase and prolongs the life-span of cells.No effect of hTERT gene on some type of differentiation potential of MSCs is present.
